Chiral two-phase system and method for resolution of racemic mixtures and separation of diastereomers

ABSTRACT

A chiral two-phase system for resolution of racemic mixtures, or for separation of diastereomers, is disclosed. The system comprises two immiscible liquid phases and one or more enantioselectively binding chiral components, each of which is substantially in one of said phases. 
     Also disclosed is a method for chiral resolution of racemic mixtures, or for separation of diastereomers. Use is here made of the fact that different enantiomers are partitioned differently between the phases in the above-mentioned two-phase system in that one of the enantiomers is selectively bound to one of the chiral components which is substantially in one of said phases.

The invention relates to a system and a method for chiral resolution ofracemic mixtures and for separation of diastereomers.

The pharmaceutical industry generally makes great demands on the opticalpurity of the pharmaceutical preparations. However, the synthesis of thedrug frequently results in a relatively low optical purity, and theresulting product must be enriched by the right enantiomer by somemethod of separation.

An attractive method in the context is the direct chiral resolutionbased on HPLC technique (see for example reference 1). However, becauseof the low capacity of these systems, the use is restricted to theanalytical scale, and therefore there is need in the art for a systemwhich is suitable for upscaling and can be used for preparativepurposes.

The invention aims at providing a chiral two-phase system for resolutionof racemic mixtures, or for separation of diastereomers, said systembeing characterised in that it comprises two immiscible liquid phasesand one or more enantioselectively binding chiral components, each ofwhich is substantially in one of said phases.

Furthermore, the invention aims at providing a method for chiralresolution of racemic mixtures, or for separation of diastereomers, saidmethod being characterised in that the different enantiomers ordiastereomers are partitioned differently between the phases of a chiraltwo-phase system comprising two immiscible liquid phases and one or moreenantioselectively binding chiral components, each of which issubstantially in one of said phases, by selectively binding one of saidenantiomers or diastereomers to one of the chiral components.

The two-phase system according to the invention thus is aliquid/liquid/two-phase system for direct chiral resolution. The phasesystem consists of two immiscible liquid phases which may consist eitherof two immiscible solvents or of two different polymers mixed withwater. Examples of immiscible solvents are water and butanol. Examplesof different polymers which in mixture with water provide two immisciblephases are a polysaccharide (such as dextran, Aquaphase) and apolyalcohol (polyethylene glycol=PEG).

Phase systems of this type are capable of resolving chiral components,such as proteins, carbohydrates, crownethers and amino acids, orderivatives thereof. By selecting suitable conditions in the two-phasesystem, such a chiral component can be placed to almost 100% in onephase, whereas other components partition themselves more equallybetween the two phases.

By selecting the chiral component such that it selectively binds oneenantiomer in a racemic mixture or one component in a mixture ofdiastereomers, this enantiomer will lie substantially within one phase.

After that, the two phases may be separated either by extraction or bymeans of a multistage process, such as countercurrent distribution.

Examples of carbohydrates that may be used as chiral components in thesystem according to the invention are cyclodextrin and cellulose. Aschiral amino acids, use may be made of D- and/or L-proline, and in thatcase one phase may contain a D-proline derivative and the other phase aL-proline derivative.

The invention will now be described in more detail, reference being hadto the following nonrestrictive Examples.

EXAMPLE 1

The protein BSA (bovine serum albumin) is used as chiral component forseparation of D,L-tryptophan. The protein was located in the bottomphase of a PEG/dextran system.

Chemicals and phase system

Dextran 40 was obtained from Pharmacia (Uppsala, Sweden), andpolyethylene glycol PEG 6000 (now renamed PEG 8000) from Union Carbide(N.Y., USA). The composition of the phase system was: 10% (w/w) Dextran40, 7% (w/w) PEG 6000, 0.1 M sodium chloride and 50 mM sodium carbonatebuffer (pH 9.2). Bovine serum albumin (6.5 g) (Sigma, No. A-3912,fraction V) was added per 100 g phase system. The albumin-containingphase system was shaken carefully and then left overnight in cold store(4° C.). D- and L-tryptophan had been obtained from Sigma and L-(sidechain-2,3-³ H)-tryptophan, specific activity: 50 μCi per nmol, fromAmersham International (U.K.).

Countercurrent distribution

An automatic countercurrent distribution equipment with 60 cavities wasused (5, 6). To cavities Nos. 1-59, 0.79 ml of respectively top andbottom phase was added, and cavity No. 0 was filled with 0.79 ml bottomphase and the sample is (9 μmol of each enantiomer of tryptophan)dissolved in 0.79 ml top phase. The shaking time was 40 s, and thepartition time 12 min. After 60 transfers (4° C.) the contents of thecavities were collected in a fraction collector. The top phase of everythird fraction was diluted to give a suitable absorbance at 280 nm. Theradioactivity both in the top phase and in the total system was measuredin a beta-scintillation counter. The scintillation liquid was Lumagel(Lumac, 6372 AD Schaesberg, The Netherlands).

Measuring the binding of L-tryptophan to serum albumin

In fractions Nos. 0-5 the radioactivity of the top phase and of thetotal phase system was measured, and the difference therebetween gavethe radioactivity of the bottom phase. From the partition coefficient ofL-tryptophan, the amount of free L-tryptophan in the bottom phase wascalculated. Since the total radioactivity of the bottom phase was known,the amount of L-tryptophan bound to serum albumin could be calculated(for further details, see FIG. 2 in reference 7).

Results and discussion

The partition of serum albumin depends on, inter alia, the molecularweight of the polymers and the ion content. Here, a phase system wassought in which serum albumin has a low partition coefficient, and thisis made possible with dextran of low molecular weight (8) and sodiumchloride as the dominating salt. In this system, 95% of the serumalbumin were in the bottom phase, and the free tryptophan partitionedmore equally between the phases (partition coefficient: 1.2).

When the enantiomers were applied separately in the countercurrentdistribution, the profiles according to FIG. 1 were obtained. Theenantioselectivity is obvious: the L-enantiomer was retained more in thebottom phase than the D-enantiomer (G_(L) =0.13,G_(D=) 0.39). Aseparation factor (G_(D) /G_(L)) of 3.1 was obtained. It shouldtherefore be possible to obtain several optically pure fractions.

Similar results were obtained on application of the racemic mixture(FIG. 2). No resolution of the racemate was obtained when separation wascarried out without serum albumin. A minor proportion oftritium-labelled L-tryptophan was added, and the major proportionresulted in a peak in the same location as the unlabelled L-formaccording to FIG. 1. However, part of the tritium labelling was found infractions Nos. 20-40, presumably because of labelled impurities(degradation products) in the tritium-labelled L-tryptophan which wasemployed and which was 2 years old.

By determining the partition of the tritium-labelled L-tryptophan infractions Nos. 0-5, the concentration of free and bound L-tryptophan inthe bottom phase, containing serum albumin, could be calculated. AScatchard plot of these data is shown in FIG. 3. An association constantof 2.9·10⁴ M⁻¹ was obtained, which is well in agreement with publishedvalues obtained by other methods (2, 9-12). The number of binding siteswas 0.4 (i.e. less than 1), which is to be expected according to otherstudies (2, 10-12). The low number of binding sites in some studies maybe due to the fact that fatty acids are present in the BSA preparation.Since the serum albumin in this study was practically free from fattyacids, a more likely explanation is an inhibition by the phase systempolymers. Polyethylene glycol resembles decanol which has been found toreduce the binding of tryptophan to bovine serum albumin (2).

EXAMPLE 2

As chiral component, use is made of cellulose in solid form which waslocated in the bottom phase of a dextran-PEG-phase system and used forseparation of D,L-tryptophan.

Chemical and phase system

Dextran 40 (Pharmacia) and Poly(ethylene glycol) PEG 8000 (UnionCarbide, N.Y.). The composition of the phase system was: 10% (w/w)Dextran, 7% (w/w) PEG, 0.5% (w/w) Imidazole and 0.1 M sodium citrate, pH8.0. 7.5 g cellulose were added per 100 g phase system. The phase systemwas shaken carefully and then left overnight in cold store (4 C.). D-and L-tryptophan had been obtained from Sigma.

Countercurrent distribution

A 60-cavity automatic countercurrent distribution equipment was used(5). To cavities Nos. 1-59, 0.79 ml of respectively top and bottom phasewas added. Cavity No. 0 was filled with 0.79 ml bottom phase and thesample (0.40 μmol of each enantiomer) dissolved in 0.79 ml top phase.Shaking was conducted for 40 s, and the partition time was 20 min. After60 transfers in cold store, the contents of each cavity were collectedin 60 test tubes.

Analysis and results

The absorbance at 280 nm on every other top phase was measured directly(FIG. 4).

EXAMPLE 3

As chiral component, use was made of the protein BSA (bovine serumalbumin) in an Aquaphase-PEG-phase system for separation ofR,S-methylsulfinyl benzoic acid on a semipreparative scale.

Chemicals and phase system

Aquaphase (Perstorp) and Poly(ethylene glycol)PEG 8000 (Union Carbide,New York). The composition of the phase system was: 14% (w/w) Aquaphase,5% (w/w) PEG, 40 mM NaCl and 20 mM sodium phosphate, pH 5.3. 7.0 gbovine serum albumin (Sigma) were added per 100 g phase system. Thephase system was shaken carefully and then left overnight in cold store(4° C.).

Countercurrent distribution

Use was made of an automatic countercurrent distribution equipmentcomprising 60 cavities (5). To cavities Nos. 1-59, 0.79 ml ofrespectively top and bottom phase was added. The cavity No. 0 was filledwith 0.79 ml bottom phase and the sample (R,S-methylsulfinyl benzoicacid, 17 mg, 92 μmol) dissolved in 0.79 ml top phase. Shaking wasconducted for 40 s, and the partition time was 20 min. After 60transfers in cold store, the contents of each cavity were collected in60 test tubes.

Analysis and results

Part of the top phase in every other tube was diluted to a suitableabsorbance 225 nm (FIG. 5). 0.3 ml of the top phase of fractions Nos.10-13 was combined and diluted with part of 40 mM NaCl, 20 mM sodiumphosphate, pH 5.3, whereupon the optical rotation was measured in aPerkin-Elmer 141 polarimeter. Fractions Nos. 14-17, 18-20, 21-24, 25-27,28-30 and 31-33 were combined, diluted and measured in the same manner.The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                        22                                                                            α D                                                     ______________________________________                                        Fractions Nos. 10-13                                                                            4.30° C.                                             Fractions Nos. 14-7                                                                             3.80° C.                                             Fractions Nos. 18-20                                                                            3.85° C.                                             Fractions Nos. 21-24                                                                            4.48° C.                                             Fractions Nos. 25-27                                                                            4.02° C.                                             Fractions Nos. 28-30                                                                            4.19° C.                                             Fractions Nos. 31-33                                                                            4.22° C.                                             ______________________________________                                    

EXAMPLE 4

As chiral component, use was made of beta-cyclodextrin coupled toAquaphase (Perstorp) which was partitioned in an Aquaphase/PEG-phasesystem for separation of R,S-terbutaline.

Chemicals and phase system

Aquaphase (Perstorp), Poly(ethylene glycol)PEG 8000 (Union Carbide,N.Y.), p-toluene sulfonyl chloride (Sigma), 1,4-diamino butane (Janssen,Belgium), β-cyclodextrin (Stadex AB, Sweden).

Synthesis of p-toluene sulfonyl-β-cyclodextrin(β-CD-OTs)

25 g (22 mmol) β-cyclodextrin (washed with diethyl ether and dried overP₂ O₅) are dissolved in 100 ml dry pyridine, the solution is saturatedwith N₂ and placed in an ice bath. 4.18 g (22 mmol) p-toluenesulfonylchloride are dissolved in 20 ml dry pyridine, the solution is saturatedwith N₂ and placed in an ice bath. The two solutions are mixed andcaused to react at room temperature overnight. The solution isevaporated, and the resulting tough oil is recrystallised from distilledwater. The product is dried over P₂ O₅. Yield 25.9 g. Elementaryanalysis gave C: 36.9-37.1%, H: 5.36-5.37%, N: 1.06-1.08%, S: 0.38%.Thus, the product contains 6.95% (w/w) pyridine, and about every sixthβ-cyclodextrin unit is tosylated. R_(f) 0.80 (Silicaplatten, Merck inCHCl₃ -MeOH 9-1).

Synthesis of p-toluene sulfonyl-Aquaphase(Aqph-OTs)

10 g (61 mmol) Aquaphase (dried over P₂ O₅) are slurried in 75 ml drypyridine, the mixture is saturated with N₂ and placed in an ice bath. 22g (116 mmol) p-toluenesulfonyl chloride are dissolved in 75 ml drypyridine, the solution is saturated with N₂ and placed in an ice bath.The components are mixed, and the mixture is slowly agitated at roomtemperature for 3 days. A minor undissolved residue remains which isfiltered off, whereupon the solution is evaporated. The resultingsemicrystalline mass is treated with 500 ml 0.5 M sodium phosphatebuffer, pH 7, for some hours, whereupon the product is obtained. Thesubstance is washed carefully with distilled water and then dried overP₂ O₅. Yield 16 g. Elementary analysis gave C: 28.2-28.5%, H:2.81-2.88%, N: 0.70-0.72%, S: 6.01-6.16%. (Elementary analysis ofAquaphase gave C: 42.6-42.8%, H: 6.60-6.65.) The product thus contains4.61% (w/w) pyridine, and every other carbohydrate monomer is tosylated.R_(f) 0.00-0.10, no traces of p-toluenesulfonyl chloride (Silicaplatten,Merck in CHCl₃ -MeOH 9-1 and CHCl₃ -MeOH-HOAc-H₂ O 6-4-1-1).

Synthesis of 1,4-diamino-butane -Aquaphase(H₂ N-(CH₂)₄ -NH-Aqph)

15 g (60 mmol) Aqph-OTs (above) are added in batches to 60 ml1,4-diamino butane. After the addition, the solution is heated to 80° C.for 4 hours and is then left overnight at room temperature. The solutionis evaporated, the remainder is diluted with 150 ml distilled water, andpH is adjusted to 7 with 6 M HCl. The neutralised solution is dialysedagainst 3×20 1 distilled water. Freeze drying gave 2.6 g product.Elementary analysis gave C: 43.2-43.3%, H: 6.85-6.97%, N:7.28-7.59%.Thus, every other carbohydrate monomer has been provided withan amino spacer. R_(f) 0.00-0.10 (Silicaplatten,Merck in CHCl₃ -MeOH 9-1and CHCl₃ -MeOH-HOAc-H₂ O 6-4-1-1), no traces of 1,4-diamino butane.

Synthesis of β-CD- NH-(CH₂)₄ -NH-Aqph

2,6 g (13 mmol) H₂ N-(CH₂)₄ -NH-Aqph (above) are dissolved together with15 g (actually 12 mmol tosylated β-cyclo dextrin) β-CD-OTs (above)containing 11 mmol pyridine and 95 ml DMF. After agitation at roomtemperature overnight, 0.8 ml triethyl amine is added. Coupling isallowed to continue for 6 days at room temperature, and finally heatingis effected to 80° C. during 4 hours. The solution is evaporated, andthe remainder is diluted with 50 ml distilled water, pH is adjusted with1 M HCl to 7. The solution is dialysed against 3×20 1 distilled water.Freeze drying gave 3.9 g product. Elementary analysis gave C:42.3-42.6%, H: 6.26-6.27%, N: 2.24-2.32%. Thus, at least every thirdcarbohydrate monomer has been provided with a β-cyclodextrin unit(actually 0.4 mol β-cyclodextrin units/mol carbohydrate monomer). R_(f)0.00-0.10 (Silicaplatten, Merck in CHCl₃ -MeOH 9- 1 and CHCl₃-MeOH-HOAc-H₂ O 6-4-1-1), ninhydrin positive reaction left. Compositionof the phase system: 14% (w/w) Aquaphase, 2.5% (w/w) β-CD-NH-(CH₂) ₄-NH-Aqph, 5% PEG, 0.1 M Li₂ SO₄, 6.25 mM sodium citrate, pH 6.0. Thephase system was shaken carefully and left overnight in cold store (4°C.).

Countercurrent distribution

Use was made of an automatic countercurrent distribution equipment with60 cavities (5). To cavities Nos. 1-59, 0.79 ml of respectively bottomand top phase was added, cavity No. 0 was filled with 0.79 ml bottomphase and the sample (4.5 μmol R,S-terbutaline) dissolved in 0.79 ml topphase. Shaking was conducted for 40 s, and the distribution time was 20min. After 60 transfers in cold store, the contents of the respectivecavities were collected in 60 test tubes.

Analysis and results

The absorbance at 280 nm on the top phase of every third fraction wasmeasured (FIG. 6).

EXAMPLE 5

As chiral component, use was made of L-proline (Pro) coupled toAquaphase (Perstorp) which was partitioned in an Aquaphase/PEG-phasesystem and used for separation of D,L-tryptophan.

Chemicals and phase system

Aquaphase (Perstorp), Polyethylene glycol PEG 8000 (Union Carbide,N.Y.), p-toluene sulfonyl chloride (Sigma), Proline (Sigma). Compositionof the phase system: 5% (w/w) Aquaphase-Pro, 10% (w/w) Aquaphase, 5%(w/w) PEG 8000, 0.1 M NaCl.

Synthesis of p-toluene sulfonyl-Aquaphase (Aqph-OTs)

10 g (61 mmol) Aquaphase (dried over P₂ O₅) is slurried in 75 ml drypyridine, the mixture is saturated with N₂ and placed in an ice bath. 22g (116 mmol) p-toluene sulfonyl chloride are dissolved in 75 ml drypyridine, the solution is saturated with N₂ and placed in an ice bath.The components are mixed, and the mixture is agitated slowly at roomtemperature for 3 days. A minor undissolved residue remains which isfiltered off, and the solution is evaporated. The resultingsemicrystalline mass is treated with 500 ml 0.5 M sodium phosphatebuffer, pH 7, for several hours during which the product is obtained.The substance is washed carefully with distilled water and then driedover P₂ O₅. Yield 16 g. Elementary analysis gave C: 28.2-28.5%, H:2.81-2.88%, N: 0.70-0.72%, S: 6.01-6.16%. Elementary analysis ofAquaphase gave C: 42.6-42.8%, H: 6.60-6.65%). Thus, the product contains4.61% (w/w) pyridine. Every other carbohydrate monomer is tosylated.R_(f) 0.00-0.10, no traces of p-toluene sulfonyl chloride(Silicaplatten, Merck in CHCl₃ -MeOH 9-1 and CHCl₃ -MeOH-HOAc-H₂ O6-4-1-1).

Synthesis of Aquaphase-Pro

10 g (40 mmol) Aqph-OTs and 7 g (60 mmol) L-proline are slurried in 100ml DMF under nitrogen gas. 6 g triethyl amine are added, and the mixtureis heated to 80° C. for 10 hours, whereupon it is left overnight at roomtemperature. After filtration and evaporation, the substance isdissolved in 150 ml water and neutralised, followed by freeze drying.The product is dried over P₂ O₅. R_(f) 0.4 (Silicaplatten, Merck inCHCl₃ -MeOH-HOAc-H₂ O: 6-4-1-1). Positive ninhydrin reaction.

Countercurrent distribution

Use was made of an automatic countercurrent distribution equipmenthaving 60 cavities (5). To cavities Nos. 1-59, 0.79 ml of respectivelybottom and top phase was added, cavity No. 0 was filled with 0.79 mlbottom phase and the sample (5 mg D,L-tryptophan) dissolved in 0.79 mltop phase. Shaking was conducted for 40 s, and the distribution time was20 min. After 60 transfers in cold store, the respective cavity contentswere collected in 60 test tubes.

Analysis and results

The absorbance at 280 nm on the top phase of every third fraction wasmeasured (FIG. 7).

EXAMPLE 6

In modification of Example 5, D-proline coupled to PEG was added to thePEG phase. Thus, L-proline-Aquaphase is in the bottom phase, andD-proline-PEG in the top phase. Due to opposed selectivity, there isthus obtained a more efficient separation if Example 5 is followed inother respects. D-proline-PEG is simply coupled to PEG via tosyl ortresyl activation.

Our results indicate that liquid/liquid/two-phase systems may be usedfor direct chiral resolution of racemic mixtures if anenantioselectively binding component is included in the phase system.These systems may be used both for analytical and for preparativepurposes. Since liquid/liquid distribution is readily upscaled, thesephase systems are especially interesting for large scale resolution ofracemic mixtures.

FIG. 1 shows countercurrent distribution of L-tryptophan () andD-tryptophan () applied separately (9 μmol.). The absorbance in the topphase of every third fraction was measured at 280 nm.

FIG. 2 shows countercurrent distribution of racemic L,D-tryptophan (18μmol), with traces of (³ H)-L-tryptophan. The absorbance in the topphase of every third tube was measured at 280 nm. To another set ofevery third tube, 1.0 ml distilled water was added to give a one-phasesystem from which samples were taken for estimation of (³H)-L-tryptophan content. Radioactivity is given in percent of totalamount of radioactivity added.

FIG. 3 is Scatchard plot of binding between L-tryptophan and bovineserum albumin, calculated on values from tubes Nos. 0-5.

FIG. 4 shows countercurrent distribution of L-tryptophan () andD-tryptophan () applied separately in the equipment. Separation factor(G_(D) /G_(L)) 1.23.

FIG. 5 shows countercurrent distribution of R,S-methylsulfinyl benzoicacid. Separation factor 1.56.

FIG. 6 shows countercurrent distribution of R,S-tetrabutaline(R,S-[3,5-dihydroxyphenyl]-2-[tert(-butylamino]ethanol).

FIG. 7 shows countercurrent distribution of D,L-tryptophan in aPro-Aquaphase/Aquaphase/PEG system.

REFERENCES

1. S. Allenmark, J. Biochem. Biophys. Meth., 9 (1984) 1-25.

2. P.-Å. Albertsson, Partition of Cell Particles and Macromolecules,Wiley, N.Y., 1971.

3. P.-Å. Albertsson, B. Andersson, C. Larsson and H.-E. Åkerlund, Meth.Biochem. Anal., 28 (1982) 115-150.

4. P.-Å. Albertsson, Meth. Biochem. Anal., 29 (1983) 1-24.

5. V.P. Shanbhag, R. Sodergå, H. Carstensen and

P.-Å. Albertsson, J. Ster. Biochem., 4 (1973)

537.

6. L. Backman, J. Chromatogr., 196 (1980) 207-216.

7. G.F. Fairclough, Jr and J.S. Fruton, Biochemistry, 5 (1966) 673-683.

8. T.P. King and M. Spencer, J. Biol. Chem., 245 (1970) 6134-6148.

9. V.J. Cunningham, L. Hay and H.B. Stoner, Biochem., J., 146 (1975)653-658.

We claim:
 1. A method for chiral resolution of racemic mixtures, or forseparation of diastereomers, wherein the different enantiomers ordiastereomers are partitioned differently between the phases of a chiraltwo-phase system comprising two immiscible liquid phases and one or moreenantioselectively binding chiral components, each of which issubstantially in one of said phases, by selectively binding one of saidenantiomers or diastereomers to one of the chiral components.
 2. Amethod according to claim 1, wherein the two immiscible liquid phasescomprise two different polymers.
 3. A method according to claim 2,wherein one of the two immiscible liquid phases comprises a polyalcoholand the other of the two immiscible liquid phases comprises apolysaccharide.
 4. A method according to claim 3, wherein thepolyalcohol comprises polyethylene glycol.
 5. A method according toclaim 3, wherein the polysaccharide comprises dextran.
 6. A methodaccording to claim 1, wherein the two immiscible liquid phases comprisetwo immiscible solvents.
 7. A method according to claim 6, wherein oneof the two immiscible solvents comprises water and the other of the twoimmiscible solvents comprises butanol.
 8. A method according to claim 1,wherein the one or more enantioselectively binding chiral components areselected from the group consisting of protein, carbohydrate, amino acid,and derivatives thereof.
 9. A method according to claim 8, wherein theprotein comprises albumin.
 10. A method according to claim 8, whereinthe carbohydrate is selected from the group consisting of cyclodextrin,cellulose, and derivatives thereof.
 11. A method according to claim 8,wherein the amino acid is selected from the group consisting ofD-proline, L-proline, and derivatives thereof.
 12. A method according toclaim 11, wherein D-proline or a derivative thereof is substantially inone of the two immiscible liquid phases, and L-proline or a derivativethereof is substantially in the other of the two immiscible liquidphases.
 13. A method according to claim 1, further comprising separatingthe two immiscible liquid phases by countercurrent distribution.
 14. Amethod for chiral resolution of a mixture of a D-tryptophan enantiomerand a L-tryptophan enantiomer, wherein the enantiomers are partitioneddifferently between phases of a chiral two-phase system comprising:(a)two immiscible liquid phases, including a dextran liquid phase and apolyethylene glycol liquid phase; and (b) bovine serum albumin, whereinthe albumin is substantially in one of the liquid phases;by selectivelybinding the L-tryptophan enantiomer to the bovine serum albumin.